The present invention relates in general to supported fluid bilayers and methods of confining them to selected areas. More specifically, the invention relates to microfabricated arrays of independently-addressable supported fluid bilayer membranes and their uses.
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Over the last several years, a number of high-throughput screening methods have been developed to facilitate the screening of thousands, if not millions, of compounds for a desired activity or activities. Such methods are typically based on detecting the binding of a potentially effective compound to a receptor. While these binding assays are effective at constraining the universe of compounds which may have the desired activity, they are typically not well-suited for evaluating this activity with any degree of detail.
The biological activity of potentially active compounds is typically evaluated using less efficient but more informative xe2x80x9csecondary screensxe2x80x9d or assays which typically require a substantial input of time by a trained technician or scientist. For evaluation of candidate compounds affecting integral membrane proteins such as receptors and ion channels, the amount of time required per compound may be several hours or days if the assay includes effects on electrophysiological activity. Accordingly, there is a need for a more efficient xe2x80x9csecondary screenxe2x80x9d of compounds affecting the activity of such integral membrane proteins, to identify those few compounds that justify further detailed analysis.
In one aspect, the present invention includes a surface detector array device. The device includes a substrate having a surface defining a plurality of distinct bilayer-compatible surface regions separated by one or more bilayer barrier regions, a bulk aqueous phase covering the substrate surface, a lipid bilayer expanse carried on each of the bilayer-compatible surface regions, and an aqueous film interposed between each bilayer-compatible surface region and corresponding lipid bilayer expanse. In a general preferred embodiment, the bilayer-compatible surface regions and the bilayer barrier surface regions are formed of different materials.
The bilayer-compatible surface region may be formed from any of a variety of materials having such bilayer-compatible surface properties, including SiO2, MgF2, CaF2, and mica, as well as a polymer film, such as a polyacrylamide or dextran film. SiO2 is a particularly effective material for the formation of a bilayer-compatible surface region.
The bilayer barrier surface region may be formed from any of a variety of materials having such bilayer barrier surface properties, including gold, positive photoresist and aluminum oxide.
In a general embodiment, the lipid bilayer expanse contains at least one lipid selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol, and sphingomyelin.
In one embodiment, the device contains between about 10 and about 100 distinct bilayer-compatible surface regions. In another embodiment, the device contains at least about 2500 distinct bilayer-compatible surface regions. In yet another embodiment, the device contains at least about 25,000 distinct bilayer-compatible surface regions. In still another embodiment, the device contains at least about 2.5 million distinct bilayer-compatible surface regions.
The bilayer-compatible surface regions are separated from one another, in one general embodiment, by bilayer barrier regions that are between about 1 xcexcm and about 10 xcexcm in width.
The lipid bilayer expanses on different bilayer-compatible surface regions may have different compositions, and may further include a selected biomolecule, with different expanses having a different biomolecule, such as transmembrane receptor or ion channel. The biomolecule may be covalently or non-covalently attached to a lipid molecule. Examples of non-covalent interactions include electrostatic and specific molecular interactions, such as biotin/streptavidin interactions. Examples of biomolecules include proteins, such as ligands and receptors, as well as polynucleotides and other organic compounds.
In another aspect, the invention includes a method of forming a surface detector device having a plurality of independently-addressable lipid bilayer regions. The method includes the steps of (i) treating a planar substrate to form a substrate surface defining a plurality of distinct bilayer-compatible surface regions separated by one or more bilayer barrier regions, and (ii) applying a suspension of lipid bilayer vesicles to the plurality of distinct bilayer-compatible surface regions under conditions favorable to the formation of supported bilayers on the bilayer-compatible surface regions. The applying then results in the formation of supported bilayer membranes on the bilayer-compatible surface regions.
In yet another aspect, the invention includes a method for detecting a selected ligand in a mixture of ligands. The method includes the steps of (i) contacting the mixture with a biosensor surface detector array device such as described above, and (ii) detecting binding of the selected ligand to receptors which specifically bind it.
In still another aspect, the invention includes a surface detection array device for use in a biosensor. Such a device includes (i) a substrate having a surface defining a plurality of distinct bilayer-compatible surface regions separated by one or more bilayer barrier regions, (ii) a bulk aqueous phase covering the substrate surface, (iii) a lipid bilayer expanse carried on each of the bilayer-compatible surface regions, and (iv) an aqueous film interposed between each bilayer-compatible surface region and corresponding lipid bilayer expanse. Each bilayer expanse contains a species of receptor or biomolecule, and different bilayer expanses contain different species of receptors or biomolecules.
Another aspect of the present invention provides for a surface detector array device, comprising a substrate having a surface defining a plurality of distinct bilayer-compatible surface regions separated by one or more bilayer barrier regions, a bulk aqueous phase covering said substrate surface, a lipid bilayer expanse carried on each of said bilayer-compatible surface regions, and an aqueous film interposed between each bilayer-compatible surface region and corresponding lipid bilayer expanse, wherein said bilayer-compatible surface regions and said bilayer barrier surface regions are formed of different materials, and wherein each bilayer-expanse carried on each bilayer-compatible region is compositionally different than adjacent bilayer-expanses. Other embodiments of the invention further include a plurality of groups of said bilayer-compatible regions, wherein said groups each define an area where said bilayer-expanses are compositionally similar, and where the bilayer-expanses within different groups are compositionally different.
The invention further provides a method for forming an array of biosensor regions, where each region has a different, known lipid bilayer composition comprising the steps of:
providing a biosensor array having a plurality of lipid bilayer compatible regions, each compatible region being surrounded by one or more bilayer barrier regions,
providing a gradient forming devices loaded with two or more different lipid bilayer compositions, the gradient forming device in fluid communication with a spot forming device for forming spots on a surface,
providing a multi-axis translation table for holding and translating a biosensor array workpiece,
placing a biosensor array workpiece that has a plurality of bilayer compatible regions surrounded by one or more barrier regions, and
forming spots of mixed lipid bilayer compositions resulting from the gradient forming device forming a gradient and translating the table in at least one axis while dispensing such composition mixture as it is formed thereby dispensing to different, consecutive locations different ratios of each lipid bilayer compositions.
The invention further provides a method for making gradient biosensor array comprising the steps of:
mixing together first and second different lipid bilayer forming compositions contained from first and second sources by flowing in a substantially laminar flow, two different compositions from two different sources into one mixing chamber that substantially retains the laminar flow character of the two different compositions while flowing through the mixing chamber, where the facing edges of each different composition mix to form a gradient having a first edge and a second edge and further comprising composition combinations of different ratios beginning from the first edge of the gradient that faces the first composition, and ending at the second edge of the gradient that faces the other, second composition, and where the mixing chamber is adapted to dispense the gradient in a substantially laminar flow across the surface of the array, and
where the compositions contained in the gradient are captured and retained upon initial contact by bilayer-compatible regions of the array.
These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying drawings.